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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Gene Expression of Ovarian Steroidogenic EnzymesN. Saito, K. Shimada Department of Animal Physiology, Faculty of Agricultural
Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan There are several hierarchical follicles in the ovary of the laying hen. During the period of follicular growth, the profile of steroid hormone production in isolated granulosa and theca layers has been well defined. We have found estradiol (E) levels were highest in the theca interna and externa of the smaller follicles, decreasing progressively with follicular growth. Testosterone (T) levels in the theca interna of less mature (F2F4) follicles were greater than in the most mature (F1) follicle, whereas theca externa contained little T. mRNA levels of P45017ahydroxylase (P45017a) were high in the theca interna of F2F3 follicles, whereas the P450aromatase (P450arom) mRNA levels were high in theca externa of the F3F4 follicles. These specific changes of mRNA levels reflected the pattern of E in this compartment. Either mRNA levels of P45017a and P450arom were extremely low in the granulosa layers during the period of follicular growth. In conclusion, E and T synthesis in the chicken ovary is regulated, at least in part, by mRNA levels of P45017a and P450arom in the theca interna and theca externa layers during follicular growth. The Role of TGFa and IGF-I in Granulosa Cells during Avian Follicular MaturationO.M. Onagbesan, M.J. Peddie, I. Woolveridge, J. Williams1, M. Duclos1 Department of Physiology and Pharmacology, University of
Southampton, UK; 1INRA-Station de Recherches Avicoles, Centre de
Recherches de Tours, Nouzilly, France Our studies have established a role for growth factors in the avian ovary. Follicular growth, steroid secretion and maturation are regulated by interactions between TGFa, IGFI and gonadotrophins (LH and FSH). TGF_ is produced by theca cells whereas the major site of IGFI production is probably the granulosa. Both TGFa and IGFI stimulate granulosa cell proliferation; the effects being greater in the F3 than the F2 or F1 follicles. However, basal and LH-induced progesterone secretion is inhibited by TGFa and enhanced by IGF-1. As follicles mature from the F3 to F1 stages, the effect of TGFa decreases while that of IGF-1 increases. Ligand binding studies were performed with iodinated IGF-1 and TGFa ligands. In freshly collected granulosa homogenates, IGF-1 receptor and IGFBP (30Kda) were found. In vitro TGFa receptor number decreases with granulosa cell maturation. Culturing cells with LH or FSH increases TGFa receptor number. Separation by SDSPAGE of granulosa proteins revealed a 170Kda protein by western analysis, to which 125ITGFa could be crosslinked. This protein possesses intrinsic tyrosine kinase activity as demonstrated by 32Pincorporation into immunoprecipitated receptors or specific staining of phosphotyrosine residues. The tyrosine kinase activity was enhanced by prior TGFa exposure; TGFa also induced the formation of activated receptor dimers. The Chick Anti-Müllerian Hormone GeneD. Carré-Eusčbe, E. Oréal, N. Josso, J-Y. Picard Endocrinologie du Développement, INSERM U293, E.N.S., 1, rue
Maurice-Arnoux, 92120 Montroughe, France Müllerian duct regression in male embryos is due to early production by testicular Sertoli cells of antiMüllerian hormone (AMH), also called Müllerian inhibiting substance (MIS). AMH, cloned and studied in mammals, is a homodimeric protein of the TGFß superfamily made of two 70 kDa glycosylated chains linked by disulfide bonds. Biological activity requires a specific protein cleavage. The Cterminal domain is the active fragment, but its activity is greatly enhanced by the presence of the Nterminal domain. Mammalian AMHs are devoid of activity on chick target tissues whereas chick AMH, a 74 kDa subunit homodimer, promotes regression of Müllerian ducts and inhibition of ovarian aromatase in both mammals and birds. To gain insight into sex differentiation in birds, we have cloned the cDNA for antiMüllerian hormone, using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of crosshybridization between mammalian and chick AMH DNA. Introns were obtained by genomic PCR and the promoter cloned from a genomic DNA library. As in mammals, the chick AMH gene is composed of five exons, with identical intron positions. The protein sequences are homologous. Twelve cysteine residues and the site of cleavage are conserved. However, the chick AMH gene is larger than mammalian ones (4200 bp vs 2800bp). The AMH protein itself is larger (644 residues in chick vs 553 to 575 in mammals), due to sequence insertions, especially in the Nterminal domain. Protein sequence identity is 49% in the Cterminal domain but only 23% in the Nterminal domain. Chick AMH expression was studied by Northern blot and in situ hybridization. In the testis, AMH mRNA is present at high levels in the cytoplasm of Sertoli cells at 8 days of incubation and decreases thereafter. AMH is also expressed from 8 days of embryonic life in the left ovary and at a lower level in the right ovary, whereas in mammals ovarian AMH is only expressed in adult granulosa cells. P450scc Activity in the BrainK. Tsutsui Faculty of Integrated Arts and Sciences, Hiroshima University,
Higashi-Hiroshima 739, Japan In birds, the brain is considered to be a target site of steroids, since steroid hormones are supplied to the brain by peripheral steroidogenic glands. The avian brain is also known to be a site of steroid metabolism, such as the conversion of androgen to estrogen. However, little is known regarding steroidogenesis from cholesterol in the brain. A series of studies were therefore carried out to determine if pregrenolene (a main steroid precursor) was synthesized from cholesterol in the brain of male Japanese quail. Western immunoblot analysis with specific antibodies against the bovine cytochrome P450scc showed that an immunoreactive protein with an electrophoretic mobility similar to P450scc was present in the brain. When the mitochondrial fraction was incubated with cholesterol, pregnenolone was detected as a function of incubation time. The pregnenolone level of hypophysectomized birds was much larger in brain than in plasma. Thus, it is possible that the avian brain produces pregnenolone from cholesterol. To determine the localization of cells involved in pregnenolone synthesis, immunohistochemical analysis with the P450scc antibody was also undertaken. An intense immunoreaction was observed in cerebellar Purkinje cells. Clusters of immunoreactive cells were also detected in the hyperstriatum accessorium, ventral portions of the archistriatum and corticoid area, the preoptic area, the anterior hypothalamus and the dorsolateral thalamus. No immunoreactivity for a specific marker protein of glial cells was evident in Purkinje cells and the cells localized in the corticoid area. Thus, both neuronal and glial cells in restricted regions may possess P450scc in the brain. Neural Activation at the Onset of IncubationR.W. Lea, G.C. Georgiou, P.J. Sharp1 Department of Applied Biology, University of Central
Lancashire, Preston PR1 2HE UK; 1Division of Reproduction and
Development, Roslin Institute, Roslin, Midlothian EH25 9PS
Scotland The ring dove possesses a stereotyped breeding cycle in which two eggs are laid about 40 h apart. Both eggs hatch after 15-16 days, with both sexes participating in incubation. The onset of incubation can be predicted, beginning after the first egg and becoming firmly established after laying the second egg. Studies have been carried out to identify neural loci mediating the behavioral and neuroendocrine changes which occur at the onset of incubation. Two markers of neural activity were employed; immunocytochemistry for the presence of the nuclear protein fos and 2-deoxyglucose (2DG) autoradiography combined with quantitative image analysis. Fos-immunoreactivity was observed in cell nuclei in several areas of the forebrain, particularly the hyperstriatum, irrespective of reproductive status and no changes were seen that correlated with the onset of incubation behaviour. No fos-immunoreactivity was seen in the hypothalamus of non-breeding controls. Fos-immunoreactivity was observed in the hypothalamus in both sexes 3-4 hours after the onset of incubation in the nucleus preopticus medialis (POM) and in the caudal nucleus tuberalis (NT). Consistent with this, a significant increase in 2-DG uptake occurred in males at the onset of incubation in the POM and NT. No fos immunoreactivity was recorded in these hypothalamic areas during courtship. However, exposure of a male dove, after 7-8 days of courtship, to the sight of an incubating female for 3 h induced fos immunoreactivity in the POM. These studies identify cells in the POM and NT activated at the onset of incubation and are consistent with the suggestion that the cells in the POM are involved in the expression of incubation behaviour. (Supported by the BBSRC) Effect of Gonadotropin-Releasing Hormone on the Proliferation and Steroidogenesis of Cultured Chicken Follicular CellsL-X. Wang, Y-Q. Su College of Biological Sciences, Beijing Agricultural
University, Beijing 100094, China The present experiments were conducted to evaluate the direct effect of the chicken gonadotropin-releasing hormone(GnRH-II) on the proliferation and steroidogenesis of granulosa cells(GC) and of theca cells(TC). GC and TC were isolated from different size follicles(F1, F3, F5) of laying hens killed 1 to 3 h before ovulation and cultured in a serum-free medium. After 20 h(GC) or 36 h(TC) preculture, the cells were treated with GnRH-II or GnRH-II plus GnRH- antagonist(GA) for 4, 48 or 72 h. The levels of the steroid hormones secreted into the medium were measured by RIA The presence of the GnRH receptor on cultured cells was also quantitatively assessed by the PAP method. At the end of culture, the cell number was counted to reveal the proliferative effect of GnRH. The results showed that: (1) GnRH-II(50-500ng/ml) stimulated the proliferation of GC and TC. GA could block this function. (2)GnRH-II(100-1000ng/ml) induced the secretion of progesterone by the GC and testosterone or estradiol by the TC from both F3 and F5. This stimulation could be inhibited by GA dramatically. No effect on the TC from F1 was observed. (3) GnRH receptor immunostaining was demonstrated on the surface of cultured cells. These results indicate that GnRH-II is capable of stimulating the steroidogenesis and proliferation of chicken follicular cells by the way of a receptor-dependent mechanism. EFFECT OF PROLACTIN ON THE STEROIDOGENESIS AND PROLIFERATION OF CULTURED THECA CELLS FROM DOMESTIC HENS.Y.H. Li, C.R. Yang. Department of Animal Physiology, Beijing Agricultural
University, Beijing 100094, China. The present experiments were performed to evaluate the effect of prolactin(PRL) on the steroidogenesis and proliferation of theca cells(TC). TC were isolated from the third largest follicles of laying hens killed 16 to 18 h before ovulation, and cultured in a serum-free medium to study the function of PRL on the steroidogenesis, or cultured in a serum-free medium supplemented with insulin, EGF, cholera toxin, transferrin and selenium to evaluate the proliferating effect of PRL on theca cells. The vital functions of the cultured theca cells were proved by the response of the cells to LH stimulation. LH significantly induced the theca cells secreting testosterone(T) and estradiol (E2) into the medium in a dose-dependent manner. After 48 h preculture, in the presence of androstenedione(10-7M), the cells were treated with PRL, PRL plus FSH(100ng/ml) and PRL plus LH(50ng/ml) respectively. The results showed that: (1) PRL had an inhibitory effect on the stimulatory action of FSH and LH on theca cell function. The secretion of E2 stimulated by FSH or T and E2 induced by LH was dramatically suppressed by PRL, but PRL alone (1-1000ng/ml) had little effect on the cells secreting T and E2. (2) PRL directly stimulated mitogenesis in thecal cells. The proliferating effect of PRL was proportional to the PRL dose (1-1000ng/ml), with the maximal effect at 1000ng/ml of PRL, which resulted in a 35.5% increase in the number of cells. These results indicate that PRL stimulates the proliferation of thecal cells, although it inhibits the gonadotropin-induced steroidogenesis of theca cells. Mitogenic Effects of FGF-2 on Chicken Granulosa and theCal Cells in vitroR.D. Roberts, R.C.L. Ellis CSIRO Division of Animal Production, Locked Bag 1, Delivery
Centre, Blacktown, NSW 2148, Australia Previously we have investigated the effects of insulinlike growth factors (IGFs) and their binding proteins on DNA synthesis by cultured chicken ovarian cells. To extend our understanding to the effects of other growth factors we report here the results of experiments with fibroblast growth factor2 (FGF2). Cultures of granulosa and thecal cells were prepared from the ovaries of domestic laying hens. Once established, the cultures were treated with a range of doses of recombinant ovine FGF2. DNA synthesis in treated cells was increased 685.4% (±29.1% s.e.m.) above untreated control granulosa cell cultures (as measured by tritiated thymidine incorporation) and 518.7% (±17.6% s.e.m.) in treated thecal cell cultures compared with control thecal cell cultures. These responses were greater than the effects of either IGF1 or IGF2 on DNA synthesis in the same cell types. Unlike the IGFs, FGF2 was found to have an optimal dose for DNA synthesis (theca = 1.00 ng/ml ±0.28 s.e.m, granulosa = 0.97 ng/ml ±0.68 s.e.m.), above which DNA synthesis declined to basal levels. Binding of FGFs to their membrane receptors involves heparin sulphatelike proteoglycans (HSPGs). The heparin antagonist, hexadimethrine, will bind to cellsurface HSPGs and prevent further binding by FGFs. To study the role of HSPGs in FGFmediated mitogenesis, hexadimethrine was added to FGF2treated cultures of both granulosa and thecal cells. The results showed a dosedependent inhibition of DNA synthesis with halfmaximal inhibition occurring at 0.26 µg/ml and 0.35 µg/ml for granulosa and thecal cells, respectively; however, when hexadimethrine was added alone there was no effect on basal DNA synthesis for either cell type. Thus, FGF2 is a potent mitogen for chicken ovarian cells and this effect is apparently mediated via cellsurface HSPGs. EFFECTS OF ACTIVE IMMUNIZATION AGAINST VASOACTIVE INTESTINAL PEPTIDE (VIP) ON PROLACTIN, HYPOTHALAMIC VIP CONTENT, AND EGG PRODUCTION IN MALLARDS.C. Bluhm, M. El Halawani1. Delta Waterfowl Research Station, R. R. #1, Portage la
Prairie, MB R1N 3A1 Canada, and 1Department of Animal Science,
495 AnSci Vet Med Building, University of Minnesota, St. Paul, MN
55108, USA. Vasoactive intestinal peptide (VIP) is a prolactin (PRL) releasing factor in birds and it plays an important role in avian reproduction. Our main goal was to determine whether active immunization with VIP would suppress circulating PRL, inhibit incubation, and thereby increase egg production in captive, wild-strain mallards. Experimental hens were vaccinated with VIP conjugated to keyhole limpet hemocyanin (KLH). The first dose of the immunogen contained 125 µg VIP and was given in April 1995. Boosters were given every 4 weeks with a total of 4 immunizations. Shortly after the first immunization, hens were placed in outdoor breeding pens with a drake. Hens were checked daily for eggs and incubation activity until mid-July. Preliminary results indicate that no experimental hens incubated eggs. Total egg production was slightly greater for experimental hens compared to control hens. Data on laying patterns, clutch size, egg fertility, and egg hatchability will be related to serum prolactin levels and hypothalamic VIP content. Physiological Correlates of Reproductive Performance in Female Zebra Finches, Taeniopygia guttataT.D. Williams, M.a Mitchell1, a. Carlisle1 Department of Biological Sciences, Simon Fraser University,
Burnaby, BC V5A 1S6 Canada; 1Roslin Institute, Roslin, Edinburgh,
Scotland, EH25 9PS, UK Intraspecific variation in reproductive investment (egg size,
clutch size, laying date) is marked within avian populations, and
both the extent and systematic pattern of inter-individual
variation is maintained in captive birds breeding under
controlled environmental conditions 1. In this study, we
investigated the physiological basis of individual variation in
reproductive performance during egg production in female zebra
finches, by comparing a) egg size, F1 and F2 follicle mass, and
oviduct mass), b) body composition (pectoral muscle, heart and
liver mass), and c) plasma levels of the two main yolk
precursors, vitellogenin and very low-density lipoproteins
(triglyceride) 2. Data were obtained from non-breeding females,
and two groups of breeding females, one maintained on a low
quality diet (seed, n = 14) and the second on a high
quality diet (seed + protein, n = 12) which had previously
been shown to increase reproductive investment 3. Reproductive
investment was unrelated to liver mass; breeding females had
livers 21-26% lighter than non-breeding birds. First egg
mass and F1 follicle mass also varied independently of pectoral
muscle mass, heart mass and plasma VLDL concentrations. However,
F1 follicle mass was positively related to plasma vitellogenin
levels (r2 = 24% ). Rate of vitellogenin synthesis and/or
secretion may be more important in regulating reproductive
investment in wild birds than has been suggested by previous
studies on poultry species4. Physiological and Behavioral Consequences of Active Immunization Against Prolactin in Turkey HensS. Crisóstomo-Pinto1,2, D. Guémené1, M. Garreau-Mills1, D. Zadworny3 1Station de Recherches Avicoles, INRA-Centre de Tours, 37380
Nouzilly, France; 2Faculdade Veterinária, UECE Dpto. de Medicina
Veterinária, 60715 Fortaleza-Ce-Brasil; 3Department of Animal
Science, McGill University H9X-3V9 Canada Prolactin is widely assumed to promote incubation behavior in domestic birds and the control of its plasma concentration using physical or pharmacological treatment prevents or disrupts this behavior. This study was therefore performed to evaluate the consequences of active immunization against prolactin on incubation expression, reproductive performance and hormonal profiles. Medium White turkey hens (n=60) were injected weekly for 4 wk starting 10 wk before photostimulation and 3 times thereafter at intervals of 4-5 wk. The hens were injected i.d. with 0.5 ml of a mixture containing 100 µg of GST-tPRL (Karatzas et al., 1993), GST, oPRL or 0,9% saline in Freund's adjuvant. Results indicate that active immunization with GST-tPRL and oPRL induce the production of antibodies. The onset of egg production was unaffected but higher egg production was observed in GST-tPRL immunized birds although the difference was not statistically significant. No GST-tPRL immunized hens expressed incubation behavior whereas 20 to 30% of hens did so in the other groups. Hyperprolactinemia was detected in the GST-tPRL immunized hens starting before photostimulation and lasting until wk 10 of egg production, but not in other hens. No significant differences were observed in either plasma LH or oestradiol concentration of immunized or non immunized turkey hens. The possible presence of prolactin antibodies in the egg had no effect on the embryonic development, hatchability, growth or survival of the offspring. In conclusion, immunization against prolactin using GST-tPRL, as an immunogen, induced hyperprolactinemia, prevented incubation behavior expression, and did not affect reproductive performance or development of offspring. Prolactin Secretory Patterns and Pituitary Lactotroph Changes during the Reproductive Cycle of the Turkey HenJ.a. Proudman, B.C. Wentworth1, R. Ramesh2, W.J. Kuenzel2 Germplasm and Gamete Physiology Laboratory, Agricultural
Research Service, USDA, Beltsville, MD 20705, USA; 1Department of
Poultry Science, University of Wisconsin, Madison, WI 53706, USA;
2Department of Poultry Science, University of Maryland, College
Park, MD 20742, USA Prolactin (PRL) secretion markedly changes during the reproductive cycle of the turkey hen. A retrospective study was conducted to contrast the weekly PRL secretion patterns of high egg producers with hens which exhibited incubation behavior, and hens which became photorefractory. A second experiment compared the episodic PRL secretory patterns of laying and incubating hens, while a third study examined differences in pituitary lactotrophs and somatotrophs in laying and incubating hens. Results showed that moderate PRL levels are optimum for best egg production. Prolactin was secreted in a pulsatile pattern in both laying, incubating, and photorefractory hens, and the hyperprolactinemia associated with incubation behavior resulted from a 12-fold increase in pulse amplitude with no change in pulse frequency or duration. The area of the pituitary gland immunoreactive for PRL was 25% greater in incubating hens than in laying hens, while the area immunoreactive for GH was 45% less in incubating hens. Some lactotrophs of incubating hens were hypertrophied, and bi-hormonal cells containing both PRL and GH were observed. We suggest that hyperprolactinemia may derive primarily from a pituitary mechanism involving lactotroph proliferation and hypertrophy. This hypothesis is supported by mammalian studies which suggest that pulse frequency is controlled at the hypothalamic/neural level, while control of pulse amplitude may occur through intrapituitary changes. |